Modified Pulsatilla decoction alleviates 5-fluorouracil-induced intestinal mucositis by modulating the TLR4/MyD88/NF-κB pathway and gut microbiota

BACKGROUND Modified Pulsatilla decoction(PD),a PD with licorice and ejiao,is a classic Traditional Chinese Medicine formula with significant efficacy in treating intestinal mucositis(IM)induced by tumor therapy.However,its specific molecular and biological mechanisms remain unclear.AIM To investigate the therapeutic effect and mechanism of modified PD in IM.METHODS This study used an IM mouse model established using 5-fluorouracil injections to investigate the effects of the modified PD(3,6,and 12 g/kg)in IM.The primary chemical components of the modified PD were identified using liquid chromatography-mass spectrometry.Body weight loss,diarrhea scores,intestinal length,histopathological scores,and inflammatory cytokine levels were measured to evaluate the effects of the modified PD in IM.Effects on the TLR4/MyD88/NF-κB pathway were evaluated using western blot analysis.The intestinal microbiota was characterized using Illumina NovaSeq sequencing.RESULTS The results showed that modified PD significantly improved weight loss and diarrhea and shortened the intestines in IM mice.Mechanistically,modified PD suppressed the TLR4/MyD88/NF-κB pathway and downregulated the expression of reactive oxygen species,lipopolysaccharides,and pro-inflammatory cytokines(IL-1β,TNF-α,IFN-γ,IL-6,IL-8,and IL-17),while increasing the expression of the anti-inflammatory cytokine IL-10.Furthermore,modified PD protected the intestinal mucosal barrier by increasing the expression of tight junction proteins(occludin-1,claudin-1,and ZO-1)and mucin-2.Finally,16S rDNA sequencing revealed that modified PD improved intestinal dysbiosis.CONCLUSION Our research offers new insights into the potential mechanism of modified PD in alleviating IM and provides experimental evidence supporting its pharmaceutical application in clinical IM treatment.

Branched-chain fatty acids from goat milk alleviate ulcerative colitis via the TLR4/NF-κB/NLRP3 pathway

Branched-chain fatty acids(BCFAs)are new bioactive fatty acids with anti-inflammatory properties.However,the role of BCFAs in alleviating ulcerative colitis has not been clarified.Herein,we evaluated the protective effect of BCFAs from goat milk in mice with colitis induced using dextran sodium sulfate(DSS)and explored the corresponding mechanism.These results show that BCFAs extracted from goat milk can significantly alleviate weight loss in mice,and reduce the disease activity index and the activity of myeloperoxidase while increasing the content of antioxidant enzymes in colon tissue and reducing the oxidation stress response.These data also show that BCFAs can down-regulate the gene and protein expression of the toll-like receptor 4(TLR4)/nuclear factorκB p65(NF-κB p65)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathway,and at the same time significantly reduce the expression of pro-inflammatory factors tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β),and IL-18 in colon tissue,and significantly increase the expression of the anti-inflammatory factor IL-10.In conclusion,these results demonstrated that BCFAs in goat milk exerted effects on colitis-related inflammatory cytokines and inhibited inflammation by inducing the TLR4/NF-κB/NLRP3 pathway to alleviate DSS-induced ulcerative colitis.This study provides evidence for the potential of BCFAs as bioactive fatty acids in food products and to ameliorate ulcerative colitis development in mice.

Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates inflammatory response ulcerative colitis through TLR4/NF-κB signaling pathway

BACKGROUND Ulcer colitis(UC)is a chronic,nonspecific,and noninfectious inflammatory bowel disease.Recently,Toll-like receptors(TLRs)have been found to be closely associated with clinical inflammatory diseases.Achieving complete remission in patients with intermittent periods of activity followed by dormancy is challenging.Moreover,no study has explored the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.AIM To explore the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.METHODS This prospective clinical study included patients who met the exclusion criteria in 2020 and 2021.The patients with UC were divided into two groups(control and experimental).The peripheral blood of the experimental and control groups were collected under aseptic conditions.The expression of TLR4 protein,NF-κB,IL-6,and IL-17 was detected in the peripheral blood of patients in the experimental group and control group before and 1 month after taking the drug.Linear co rrelation analysis was used to analyze the relationship between the expression level of TLR4 protein and the expression levels of downstream signal NF-κB and inflammatory factors IL-6 and IL-17,and P<0.05 was considered statistically significant.RESULTS There were no significant differences in the patient characteristics between the control and experimental groups.The results showed that the expression levels of TLR4 and NF-κB in the experimental group were significantly lower than those in the control group(P<0.05).The levels of IL-6 and IL-17 in the experimental group were significantly lower than those in the control group(P<0.05).The TLR4 protein expression in the experimental group was positively correlated with the expression level of downstream signal NF-κB and was positively correlated with the levels of downstream inflammatory cytokines IL-6 and IL-17(r=0.823,P<0.05).CONCLUSION Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates the inflammatory response of UC through the TLR4/NF-κB signaling pathway.

The potential mechanism of Isodon suzhouensis against COVID-19 via EGFR/TLR4 pathways

Corona Virus Disease 2019(COVID-19)has brought the new challenges to scientific research.Isodon suzhouensis has good anti-inflammatory and antioxidant stress effects,which is considered as a potential treatment for COVID-19.The possibility for the treatment of COVID-19 with I.suzhouensis and its potential mechanism of action were explored by employing molecular docking and network pharmacology.Network pharmacology and molecular docking were used to screen drug targets,and lipopolysaccharide(LPS)induced RAW264.7 and NR8383 cells inflammation model was used for experimental verification.Collectively a total of 209 possible linkages against 18 chemical components from I.suzhouensis and 1194 COVID-19 related targets were selected.Among these,164 common targets were obtained from the intersection of I.suzhouensis and COVID-19.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enriched 582 function targets and 87 target proteins pathways,respectively.The results from molecular docking studies revealed that rutin,vitexin,isoquercitrin and quercetin had significant binding ability with 3 chymotrypsin like protease(3CLpro)and angiotensin converting enzyme 2(ACE2).In vitro studies showed that I.suzhouensis extract(ISE)may inhibit the activation of PI3K/Akt pathway and the expression level of downstream proinflammatory factors by inhibiting the activation of epidermal growth factor receptor(EGFR)in RAW264.7 cells induced by LPS.In addition,ISE was able to inhibit the activation of TLR4/NF-κB signaling pathway in NR8383 cells exposed to LPS.Overall,the network pharmacology and in vitro studies conclude that active components from I.suzhouensis have strong therapeutic potential against COVID-19 through multi-target,multi-pathway dimensions and can be a promising candidate against COVID-19.

Quercetin regulates depression-like behavior in CUMS rat models via TLR4/NF-κB signaling

Background:Depression is becoming increasingly prevalent around the world,imposing a substantial burden on individuals,families,as well as society.Quercetin is known to be highly effective in treating depression.However,additional research is needed to dissect the mechanisms of its anti-depressive effects.Methods:For this study,Sprague-Dawley(SD)rats were randomized into the control,model,quercetin,or fluoxetine group.The latter three groups were exposed to chronic unpredictable mild stress(CUMS)for 42 d.The first two groups received saline solution daily via oral gavage.Meanwhile,the quercetin group was orally administered a quercetin suspension(52.08 mg/kg)every day,while the fluoxetine group was orally administered a fluoxetine solution(2.08 mg/kg).Here,fluoxetine served as the positive control drug to compare the therapeutic effects of quercetin.The experimental period was 6 weeks.Depressive behaviors in rats were assessed through various physiological and behavioral measures.Additionally,pathological changes in hippocampal tissues were examined using Nissl staining.Serum cytokines were detected using an enzymelinked immunosorbent assay(ELISA),and immunohistochemistry was employed to quantify the levels and integral optical density(IOD)values of ionized calcium binding adaptor molecule-1(Iba-1)expression in the brain.Real-time fluorescence quantitative PCR(RT-qPCR)was utilized to evaluate the mRNA levels of inflammatory indicators as well as toll-like receptor 4(TLR4),and nuclear factor-κappa B P65(NF-κB P65)in hippocampus.Western blot(WB)technique was employed to observe the protein levels of TLR4,NF-κB P65,and phospho-NF-κB P65(p-NF-κB P65).Results:After 42 d of exposure to CUMS,rats exhibited a slow increase in body weight,a reduction in food intake,an abnormal preference for sugar water,and aberrant open-field behaviors.Pathological analysis revealed the disintegration,rupture,interruption,and disorganization of hippocampal neuronal cells after CUMS exposure,along with a decrease in Nissl bodies in the CA1 region.This was accompanied by the elevated expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in the serum and the upregulation of IL-1β,IL-6,and TNF-αmRNA expression in the hippocampus.Increases in Iba-1-positive cells and the IOD values of Iba-1 were detected in hippocampal microglia.Furthermore,TLR4 and NF-κB P65 mRNA and protein levels were upregulated in hippocampal tissues.Quercetin,an antidepressant,could alleviate depression-like symptoms in rats and downregulate inflammatory factors associated with the TLR4/NF-κB signaling pathway in hippocampal microglia,and its therapeutic effect was comparable to fluoxetine.Conclusion:In rat models of CUMS,quercetin may act as an antidepressant by inhibiting inflammation in hippocampal microglia via TLR4/NF-κB signaling pathway.These results offer experimental and theoretical support for applying quercetin in the clinical management of depression.

Anti-inflammation Effects of Sinomenine on Macrophages through Suppressing Activated TLR4/NF-kB Signaling Pathway

Sinomenine(SN)has been used in the clinical treatment of systemic lupus erythematosus and rheumatoid arthritis for many years.Studies showed that SN held protective effects such as anti-inflammation,scavenging free radicals and suppressing immune response in many autoimmune diseases.The purpose of the present study is to explore the mechanism of anti-inflammation of SN on lipopolysaccharide(LPS)-induced macrophages activation and investigate whether the TLR4/NF-κB signaling pathway participated in.Macrophages isolated from mouse peritoneal cavity were stimulated by 1 pg/mL LPS for 24 h.And then the cells were treated with various concentrations of SN,TLR4 inhibitor respectively for additional 48 h.Drug toxicity was detected by MTT assay and Transwell experiment was used to assess chemotaxis.Furthermore,TLR4 and MyD88 mRNA levels were detected by real-time PCR.Western blotting was used to examine TLR4,MyD88 and phosphorylated IκB protein expression in macrophages.Immunofluorescence assay was applied to observe p65 NF-κB protein expression in macrophage nucleus.We extracted macrophages with high purity and activity from the abdominal cavity of mice.SN remarkably inhibited the chemotaxis and secretion function of LPS-stimulated macrophages.It also down-regulated both the protein levels of inflammatory cytokines(TNF-α,IL-β and IL-6)and the RNA and protein levels of the key factors(TLR4,MyD88,p-IkB)in TLR4 pathway.The expression of p65 NF-κB protein in nuclei was down-regulated,which was correlated with a similar decrease in p-IκB protein level.In conclusion,SN can inhibit the LPS induced immune responses in macrophages by blocking the activated TLR4/NF-κB signaling pathway.These results may provide a therapeutic approach to regulate inflammatory responses.

β-arrestin 2 attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation in mice

AIM To study the role and the possible mechanism of β-arrestin 2 in lipopolysaccharide(LPS)-induced liver injury in vivo and in vitro.METHODS Male β-arrestin 2^(+/+) and β-arrestin 2^(-/-)C57 BL/6 J mice were used for in vivo experiments, and the mouse macrophage cell line RAW264.7 was used for in vitro experiments. The animal model was established via intraperitoneal injection of LPS or physiological sodium chloride solution. Blood samples and liver tissues were collected to analyze liver injury and levels of pro-inflammatory cytokines. Cultured cell extracts were collected to analyze the production of pro-inflammatory cytokines and expression of key molecules involved in the TLR4/NF-κB signaling pathway.RESULTS Compared with wild-type mice, the β-arrestin 2 knockout mice displayed more severe LPS-induced liver injury and significantly higher levels of proinflammatory cytokines, including interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, and IL-10. Compared with the control group, pro-inflammatory cytokines(including IL-1β, IL-6, TNF-α, and IL-10) produced by RAW264.7 cells in the β-arrestin 2 si RNA group were significantly increased at 6 h after treatment with LPS. Further, key molecules involved in the TLR4/NF-κB signaling pathway, including phosphoIκBα and phosho-p65, were upregulated.CONCLUSION β-arrestin 2 can protect liver tissue from LPS-induced injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation.

Milled flaxseed-added diets ameliorated hepatic inflammation by reducing gene expression of TLR4/NF-κB pathway and altered gut microbiota in STZ-induced type 1 diabetic mice

Flaxseed has displayed the potential beneficial as functional foods.However,most studies focused on effects of flaxseed extracts or ingredients in flaxseed.Besides,few studies showed that flaxseed extracts contributed to anti-type 1 diabetes(T1D),yet the underlying mechanism is still unknown.In the present study,16.7% of milled flaxseed(MF)-added diet was given to diabetic mice induced by streptozocin for 6 weeks.The results showed that MF feeding 1)slightly decreased blood glucose levels and improved the ability of glucose tolerance by oral glucose tolerance test,2)decreased liver tumor necrosis factor-αlevels and increased liver glycogen levels with significance via down-regulating TLR4/NF-κB pathways,3)and significantly altered some beneficial bacteria in gut microbiota.In conclusion,the present study showed that milled flaxseed showed the potential on anti-T1D through anti-inflammation via TLR4/NF-κB and altering the gut microbiota in STZ-induced diabetic mice.

Effect of dexmedetomidine on the prevention of PSH in patients with severe craniocerebral injury by regulating TLR4/My D88/NF-kappa B signaling pathway

Objective:To investigate the clinical efficacy of dexmedetomidine in the regulation of TLR4/My D88/NF-κB in the prevention of paroxysmal sympathetic over-excitation (PSH) in patients with severe head injury. Methods:One hundred patients with severe head injury who were admitted to our hospital from September 2016 to May 2019 were enrolled. The randomized digital table method was divided into 50 cases in the study group and the control group. Patients in the study group were given dexmedetomidine at a dose of 1.0 μg/kg before anesthesia induction, followed by infusion at 0.4 μg / (kg·h), and the control group was injected with the same amount of normal saline. The incidence of PSH, clinical symptoms, imaging findings, mechanical ventilation time, tracheal intubation/incision duration, ICU hospitalization time, total length of hospital stay, and GCS scores three months after discharge were compared between the two groups. At the same time, the fluorescence intensity, TLR4, NF-κB expression level and tumor necrosis factor-α (TNF-α) expression levels in peripheral blood CD14+ monocytes of the two groups were detected. Results:The incidence of PSH was significantly lower in the study group than in the control group at 7 and 3 months (P<0.05). The total length of hospital stay, duration of ICU hospitalization, intraoperative tracheotomy, and mechanical ventilation time were significantly lower in the study group than in the control group. And the GCS score was higher than the control group, and the difference was statistically significant (P<0.05). In addition, the imaging results showed that there were some differences in the location of imaging lesions between the two groups. The proportion of lesions in the ventricular system and surrounding areas was higher in the control group than in the study group (P<0.05). And the T14-T3 CD14+ PBMC MyD88 fluorescence intensity, TLR4 and NK-κB positive expression rate were significantly higher than those of T0 (P<0.05), but the MyD88 fluorescence intensity, TLR4 and NK-κB positive expression rate in the study group were significantly lower than those in the control group at T1~T3 (P<0.05). The levels of serum TNF-α in T1~T3 groups were significantly higher than those in T0 (P<0.05), but the levels of serum TNF-α in T1~T3 in the study group were significantly lower than those in the control group (P< 0.05). Conclusions:Dexmedetomidine can reduce the oxidative stress response in patients with severe head injury by inhibiting TLR4/My D88/NF-κB signaling pathway, thus effectively reducing the risk of PSH and improving the prognosis of patients.

Effects of Polysaccharides from Dicliptera chinensis(L.)Nees.on TLR4/NF-κB Signaling Pathway in Cell Model of Non-alcoholic Fatty Liver

[Objectives]To observe the effects of polysaccharides from Dicliptera chinensis(L.)Nees.on the expression of TLR/NF-κB pathway related proteins in HepG2 cells induced by oleic acid,and to explore the possible mechanism of polysaccharides from D.chinensis(L.)Nees.in the treatment of non-alcoholic fatty liver disease(NAFLD).[Methods]HepG2 cells were induced with oleic acid to establish a non-alcoholic fatty liver cell model.After intervention with 0.25 and 0.5 mg/mL of D.chinensis(L.)Nees.polysaccharides,the ALT and AST activity and TG and TC contents were detected with kits,and the changes in the expression of CDK5,TLR4,p-NF-κB and NF-κB were analyzed using Western-blotting.[Results]In the HepG2 cells induced with oleic acid,the ALT and AST activity increased significantly,the TG and TC contents increased significantly,and the expression levels of CDK5,TLR4 and p-NF-κB proteins up-regulated significantly.In the HepG2 cells intervened with D.chinensis(L.)Nees.polysaccharides,the activity of ALT and AST,the contents of TG and TC,and the expression levels of CDK5,TLR4 and p-NF-κB proteins all reduced significantly.[Conclusions]Polysaccharides from D.chinensis(L.)Nees.may interfere with NAFLD by inhibiting the TLR4/NF-κB pathway.

Bioinformatic Analysis and Experimental Verification of QJHGD on Caerulein-induced Inflammatory Response in SAP Model Rats Based on TLR4/NF-κB/My D88 Pathway

[Objectives]To conduct bioinformatic analysis and experimental verification of Qingjie Huagong Decoction(QJHGD)on caerulein-induced inflammatory response in severe acute pancreatitis(SAP)model rats based on TLR4/NF-κB/MyD88 pathway.[Methods]The effective component groups and potential targets of QJHGD were collected by the network pharmacology method.A drug-component-target network was constructed.The GO and KEGG of targets were enriched and analyzed with the aid of Metascape database,and the target pathway related to SAP inflammation was screened.The SAP rat model was established by caerulein combined with lipopolysaccharide,and QJHGD was intragastrically administered.Pancreatic tissue was observed by HE staining.In addition,enzyme-linked immunosorbent assay and immunohistochemistry were used to verify the anti-inflammatory effect of QJHGD on SAP rats and its regulatory effect on TLR4/NF-κB/MyD88 target pathway.[Results]A total of 105 active components of QJHGD and 148 key targets of SAP were predicted and screened;KEGG was enriched in 320 different pathways including toll-like receptor and NF-κB classical pathways.Animal experiment verified that QJHGD reduced serum amylase,serum lipase activity,IL-6,TNF-αlevels in SAP rats;HE staining showed the effect of QJHGD on the pathological changes of pancreas,and QJHGD inhibited the positive expression of key proteins of TLR4,NF-κB and MyD88 in the inflammatory transduction pathway.[Conclusions]The mechanism of QJHGD improving pancreatic injury in SAP rats may be related to down-regulating the expression of key proteins in the TLR4/NF-κB/MyD88 pathway.

Blautia producta displays potential probiotic properties against dextran sulfate sodium-induced colitis in mice

Blautia has attracted attention because of its potential efficacy in ameliorating host energy metabolism and inflammation.This study aims to investigate the influences of Blautia producta D4 on colitis induced by dextran sulfate sodium(DSS)and to reveal the underlying mechanisms.Results showed that B.producta D4 intervention significantly relieved body weight loss,and suppressed the elevation of pro-inflammatory cytokines(including interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β))and excessive oxidative stress(myeloperoxidease(MPO)activity,superoxide dismutase(SOD)activity,glutathione peroxidase(GSH-Px)activity,and malondialdehyde(MDA)level)in colitis mice.Moreover,the concentrations of tight junction proteins(occludin,claudin-1,and ZO-1)related to the intestinal barrier were obviously elevated,and colitis-related TLR4/NF-κB pathway activation was remarkably inhibited after B.producta D4 intervention.The intestinal microbial disorder was evidently ameliorated by increasing the relative abundance of Clostridium sensu stricto 1,Bifidobacterium,GCA-900066225,Enterorhabdus,and reducing the relative abundance of Lachnospiraceae NK4A136 group.In conclusion,oral administration of B.producta D4 could ameliorate DSS-induced colitis by suppressing inflammatory responses,maintaining the intestinal barrier,inhibiting TLR4/NF-κB pathway,and regulating intestinal microbiota balance.These results are conducive to accelerate the development of B.producta D4 as a functional probiotic for colitis.

Elaidic acid-induced intestinal barrier damage led to gut-liver axis derangement and triggered NLRP3 inflammasome in the liver of SD rats

Previous studies have shown that trans fatty acids(TFA) are associated with several chronic diseases,the gut microbiota is directly influenced by dietary components and linked to chronic diseases.Our research investigated the effects of elaidic acid(EA),a typical TFA,on the gut microbiota to understand the underlying mechanisms of TFA-related chronic diseases.16S rDNA gene sequencing on faecal samples from Sprague-Dawley rats were performed to explore the composition change of the gut microbiota by EA gavage for 4 weeks.The results showed that the intake of EA increased the abundance of well-documented harmful bacteria,such as Proteobacteria,Anaerotruncus,Oscillibacter and Desulfovibrionaceae.Plus,EA induced translocation of lipopolysaccharides(LPS) and the above pathogenic bacteria,disrupted the intestinal barrier,led to gut-liver axis derangement and TLR4 pathway activation in the liver.Overall,EA induced intestinal barrier damage and regulated TLR4-MyD88-NF-κB/MAPK pathways in the liver of SD rats,leading to the activation of NLRP3 inflammasome and inflammatory liver damage.

Salvianolic Acid B Inhibits the TLR4-NFκ B-TNFα Pathway and Attenuates Neonatal Rat Cardiomyocyte Injury Induced by Lipopolysaccharide

Objective： To investigate the role of the TLR4-NF K B-TNFa inflammation pathway on lipopolysaccharide （LPS）-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B （Sal B）. Methods： Wistar rat （1-2 days old） cardiomyocytes were isolated and cultured. Sal B 10-5mol/L, 10-6mol/L and 10-7mol/L were pro-treated for 6 h in the culture medium. LPS （1 μg/mL） was added to the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase （LDH） in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor （TNF a） and heat shock protein 70 （HSP70） in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 （TLR4） and nuclear factor kappa B （NF K B） were detected by immunohistochemistry. The mRNA expressions of TLR4 and NF K B were detected by realtime reverse transcription polymerase chain reaction （RT-PCR）. Results： （1） The concentrations of LDH and TNF a in the LPS control group were significantly higher than those in the control group （561.41 ± 67,39 U/L and 77.94± 15.08 pg/mL, versus 292.13± 26.02 U/L and 25.39 ±16.53 pg/mL, respectively, P〈0.01, P〈0.05）. Compared with the LPS control group, the concentrations of LDH and TNF α were significantly decreased in the Sal B 10-5mol/L pro-treated group （451.76 ± 83.96 U/L and 34.00± 10.38 pg/mL, respectively, P〈0.05）. （2） The TLR4 and NF K B protein expression area in the LPS control group were significantly higher than those in the control group （1712.41 ± 410.12 μm2 and 2378.15 ± 175.29 μm2, versus 418.62 ± 24.42 μ m2 and 1721.74 ± 202.87μ m2, respectively, P〈0.01）. The TLR4 and NF K B protein expression internal optical density （IOD） values in the LPS control group were also significantly higher than those in the control group （3.06 ±0.33 and 7.20± 1.04, versus 0.91 ±0.21 and 4.24±0.48, respectively, P〈0.05 and P〈0.01）. Compared with the LPS control group, the TLR4 and NF K B protein expression areas were significantly decreased in the Sal B 10Smol/L pre-treated group （1251.54± 133.82 μ m2 and 1996.37 ± 256.67 μ m2, respectively, P〈0.05）, the TLR4 and NF K B protein expression IOD values were also significantly decreased in the Sal B 10-5mol/L pretreated group （1.92 ±0.28 and 5.17 ±0.77, respectively, P〈0.05）. （3） The TLR4 and NF K B mRNA expressions （2△△CT value） in the LPS control group were significantly higher than those in the control group （3.16 ± 0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P〈0.01）. Compared with the LPS control group, the TLR4 and NF KB mRNA expressions （2△△CT value） were significantly decreased in the Sal B 10-5mol/L pre- treated group （1.34 ±0.22 and 1.74 ± 0.26, respectively, P〈0.05）. The concentration of HSP70 did not show any statistical differences in all groups （P〉0.05）. Conclusions： The TLR4-NF K B-TNF α pathway was quickly activated and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NF K B-TNF a pathway and are dose-dependent.

Modulating effects of Astragalus polysaccharide on immune disorders via gut microbiota and the TLR4/NF-κB pathway in rats with syndrome of dampness stagnancy due to spleen deficiency

The syndrome of dampness stagnancy due to spleen deficiency(DSSD)is relatively common globally.Although the pathogenesis of DSSD remains unclear,evidence has suggested that the gut microbiota might play a significant role.Radix Astragali,used as both medicine and food,exerts the effects of tonifying spleen and qi.Astragalus polysaccharide(APS)comprises a macromolecule substance extracted from the dried root of Radix Astragali,which has many pharmacological functions.However,whether APS mitigates the immune disorders underlying the DSSD syndrome via regulating gut microbiota and the relevant mechanism remains unknown.Here,we used DSSD rats induced by high-fat and low-protein(HFLP)diet plus exhaustive swimming,and found that APS of moderate molecular weight increased the body weight gain and immune organ indexes,decreased the levels of interleukin-1β(IL-1β),IL-6,and endotoxin,and suppressed the Toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)pathway.Moreover,a total of 27 critical genera were significantly enriched according to the linear discriminant analysis effect size(LEfSe).APS increased the diversity of the gut microbiota and changed its composition,such as reducing the relative abundance of Pseudoflavonifractor and Paraprevotella,and increasing that of Parasutterella,Parabacteroides,Clostridium XIVb,Oscillibacter,Butyricicoccus,and Dorea.APS also elevated the contents of short-chain fatty acids(SCFAs).Furthermore,the correlation analysis indicated that 12 critical bacteria were related to the body weight gain and immune organ indexes.In general,our study demonstrated that APS ameliorated the immune disorders in DSSD rats via modulating their gut microbiota,especially for some bacteria involving immune and inflammatory response and SCFA production,as well as the TLR4/NF-κB pathway.This study provides an insight into the function of APS as a unique potential prebiotic through exerting systemic activities in treating DSSD.

Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway

Objective:To explore the protective effect of Huoxin Pill(HXP)on acute myocardial ischemia-reperfusion(MIRI)injury in rats.Methods:Seventy-five adult SD rats were divided into the sham-operated group,model group,positive drug group(diltiazem hydrochloride,DH),high dose group(24 mg/kg,HXP-H)and low dose group(12 mg/kg,HXP-L)of Huoxin Pill(n=15 for every group)according to the complete randomization method.After 1 week of intragastric administration,the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h.Serum was separated and the levels of creatine kinase(CK),creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH),superoxide dismutase(SOD),and malondialdehyde(MDA),hypersensitive C-reactive protein(hs-CRP)and interleukin-1β(IL-1β)were measured.Myocardial ischemia rate,myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride(TTC).Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine(BATMAN)databases were used to screen for possible active compounds of HXP and their potential therapeutic targets;the results of anti-inflammatory genes associated with MIRI were obtained from GeneC ards,Drugbank,Online Mendelian Inheritance in Man(OMIM),and Therapeutic Target Datebase(TTD)databases was performed;Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment were used to analyze the intersected targets;molecular docking was performed using AutoD ock Tools.Western blot was used to detect the protein expression of Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NFκB)/NOD-like receptor protein 3(NLRP3).Results:Compared with the model group,all doses of HXP significantly reduced the levels of LDH,CK and CK-MB(P<0.05,P<0.01);HXP significantly increased serum activity of SOD(P<0.05,P<0.01);all doses of HXP significantly reduced the levels of hs-CRP and IL-1β(P<0.05,P<0.01)and the myocardial infarction rate and myocardial no-reflow rate(P<0.01).GO enrichment analysis mainly involved positive regulation of gene expression,extracellular space and identical protein binding,KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis.Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4,NFκB and NLRP3 molecules.The protein expressions of TLR4,NFκB and NLRP3 were reduced in the HXP group(P<0.01).Conclusions:HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats,and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4/NFκB/NLRP3 signaling pathway.

MANF brakes TLR4 signaling by competitively binding S100A8 with S100A9 to regulate macrophage phenotypes in hepatic fibrosis

The mesencephalic astrocyte-derived neurotrophic factor(MANF)has been recently identified as a neurotrophic factor,but its role in hepatic fibrosis is unknown.Here,we found that MANF was upregulated in the fibrotic liver tissues of the patients with chronic liver diseases and of mice treated with CCl4.MANF deficiency in either hepatocytes or hepatic mono-macrophages,particularly in hepatic mono-macrophages,clearly exacerbated hepatic fibrosis.Myeloid-specific MANF knockout increased the population of hepatic Ly6C^(high)macrophages and promoted HSCs activation.Furthermore,MANF-sufficient macrophages(from WT mice)transfusion ameliorated CCl4-induced hepatic fibrosis in myeloid cells-specific MANF knockout(MKO)mice.Mechanistically,MANF interacted with S100A8 to competitively block S100A8/A9 heterodimer formation and inhibited S100A8/A9-mediated TLR4-NF-κB signal activation.Pharmacologically,systemic administration of recombinant human MANF significantly alleviated CCl_(4)-induced hepatic fibrosis in both WT and hepatocytes-specific MANF knockout(HKO)mice.This study reveals a mechanism by which MANF targets S100A8/A9-TLR4 as a“brake”on the upstream of NF-κB pathway,which exerts an impact on macrophage differentiation and shed light on hepatic fibrosis treatment.

Whey protein peptide PEW attenuates hyperuricemia and associated renal inflammation in potassium oxonate and hypoxanthine-induced rat

Pro-Glu-Trp(PEW),a whey protein-derived peptide,has been previously shown in vitro to have antihyperuricemic potential.The current study further evaluated the roles and the underlying mechanism of PEW in the management of hyperuricemia(HUA)in rat induced by potassium oxonate(PO)and hypoxanthine.Results revealed that PEW significantly reduced the levels of uric acid(UA),creatinine(Cr),and blood urea nitrogen(BUN)in serum,and effectively suppressed the activities of xanthine oxidase(XOD)associated with UA synthesis and modulated the expression of organic ion transporters related to UA excretion.Moreover,PEW alleviated UAinduced renal inflammation by regulating oxidative stress,suppressing the level of pro-inflammatory cytokines,and inhibiting the activation of NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome and Toll-like receptor 4/myeloid differentiation factor 88/NF-kappaB(TLR4/MyD88/NF-κB)signaling pathway.Taken together,these relults indicated that PEW improved HUA and renal inflammation by inhibiting UA synthesis,promoting renal UA excretion and suppressing NLRP3 inflammasome and TLR4/MyD88/NF-κB signaling pathways.